SEED-SPECIFIC HETEROLOGOUS EXPRESSION OF A NASTURTIUM FAE
GENE IN ARABIDOPSIS RESULTS IN AN EIGHT-FOLD INCREASE IN
ERUCIC ACID CONTENT
ELZBIETA MIETKIEWSKA 1,3 , E. MICHAEL GIBLIN1 , SONG WANG1 , DENNIS L.
BARTON2 , JOAN DIRPAUL1 , VESNA KATAVIC2 , AND DAVID C. TAYLOR1
National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium
Place, Saskatoon, Saskatchewan, Canada S7N 0W9
CanAmera Foods, P.O. Box 479, 125 Willow Court, Osler, SK, S0K 3A0
Plant Breeding and Acclimatization Institute, Mlochow Research Center, Poland
A major goal of our research is to obtain, by genetic manipulation, Brassica napus L. cultivars with higher
proportions (above 66 mol%) of erucic acid (22:1) in their seed oil than in present Canadian HEA cultivars.
We have selected Tropaeloum majus, garden nasturtium, as a source of new strategic genes based on the
fact that this plant is capable of producing significant amounts of erucic acid (70-75% of total fatty acid)
and accumulates trierucin as the predominant TAG in its oil (Taylor et al. 1992).
Very long-chain fatty acids (VLCFAs) are synthesized by a membrane-bound fatty acid elongation
complex (elongase, FAE) using acyl-CoA substrates. The first reaction of elongation involves condensation
of malonyl-CoA with a long chain substrate producing a β-ketoacyl-CoA. Subsequent reactions are
reduction of β-hydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a second reduction to form the
elongated acyl-CoA. The β-ketoacyl-CoA synthase (KCS) catalyzing the condensation reaction plays a key
role in determining the chain length of fatty acid products found in seed oils and is the rate-limiting enzyme
for seed VLCFA production (Lassner et al., 1996).
Here we report isolation of a nasturtium FAE gene and demonstrate the involvement of its
encoded protein in the elongation of monounsaturated and saturated fatty acids.
2. Results and Discussion
2.1. Isolation of T. majus FAE homolog
Based on sequence homology among plant fatty acid elongase genes, a full-length cDNA clone was
amplified by PCR using a degenerate primers approach and the sequence submitted to GenBank™
(Accession number AY082610). The T. majus FAE cDNA encodes a polypeptide of 504 amino acids that is
most closely related to an FAE2 from roots of Zea mays (69 % amino acid identity; Schreiber et al., 2000),
(Figure 1). The T. majus FAE polypeptide also shared strong identity with FAEs from Limnanthes
douglasii (67%; Cahoon et al., 2000) and from seeds of jojoba (Simondsia chinensis) (63%; Lassner et al.,
1996). Homology of the nasturtium FAE to two Arabidopsis β-ketoacyl-CoA synthases AraKCS (Todd et
al., 1999) and AraCUT1 (Millar et al., 1999) involved in cuticular wax synthesis was on the level of 57%
and 53%, respectively. These homologs all exhibit the capability to elongate saturated fatty acids to
produce saturated VLCFAs. The FAE1 polypeptides involved in the synthesis of VLCFAs in Arabidopsis
(James et al., 1995) and Brassica seeds (Clemens and Kunst, 1997) showed approximately 52-54% identity
with the T. majus FAE.
50 40 30 20 10 0
Nucleotide Substitutions (x100)
Figure 1. Dendrogram of the β-ketoacyl-CoA synthase gene family based on the amino acid sequences. The alignment was carried out
by the Clustal W method using Lasergene analysis software (DNAStar, Madison, WI). The dendogram contains the sequences of the
Limnanthes (LimFAE, GenBank Acc# AF247134), jojoba (SimFAE, GenBank Acc# U37088), nasturtium (NasFAE, GenBank Acc#
AY0826190), corn (ZeaFAE, GenBank Acc# AJ292770), Arabidopsis (AraFAE, GenBank Acc# U29142; AraKCS, GenBank Acc#
AF053345: and AraCUT, GenBank Acc# AF129511) and Brassica (BraFAE, GenBank Acc# AF009563).
1.2. Tissue specific expression and copy number estimate of T. majus FAE
Northern blot analyses were performed to investigate the expression profile of the FAE gene. Total RNA
was isolated from different nasturtium tissues including roots, leaves, floral petals and mid-developing
embryos. A strong hybridization signal with FAE-specific probe was observed only with RNA isolated
from developing embryos (Figure 2A).
R L P E 1 2 3 4
Figure 2. Northern and Southern analyses of T. majus FAE.
A. Northern analysis of FAE gene expression in T.majus. Total RNA was isolated from roots (R), leaves (L), petals (P) and embryos
B. Southern blot analysis of the FAE gene in T.majus. Genomic DNA was digested with restriction enzymes: EcoRI (lane 1), AccI
(lane 2), NcoI (lane 3) and HindIII (lane 4).
A Southern blot hybridization was performed with nasturtium genomic DNA digested with several
restriction enzymes including EcoRI, AccI, NcoI and HindIII. The FAE gene has no internal EcoRI, AccI or
NcoI sites, while one internal HindIII site exists. Autoradiography revealed the presence of one strongly-
hybridizing fragment in all cases except with HindIII for which two strongly hybridizing fragments were
evident (Figure 2B). In addition a minimum of 4 weakly hybridizing fragments were detected. Thus, we
have concluded that T. majus FAE belongs to a multigenic family consisting of 4 to 6 members. A similar
multigenic family has been found for a rapeseed FAE1 gene member (Barret et al., 1998).
1.3. Seed specific expression of T. majus FAE in Arabidopsis plants
To establish the function of elongase homolog, the cDNA was introduced into Arabidopsis background
(ecotype Wasilewskija) under the control of the napin promoter. From vacuum-infiltration experiments, 25
kanamycin-resistant T1 plants were selected. The T2 progeny were collected individually from each plant
and the fatty acid composition determined. Significant changes in fatty acid composition in comparison to
the wild type (empty vector) were found. On average, the proportion of erucic acid (22:1 ∆13) increased
from 2.1% in wild type to 9.6% in T transgenic seeds at the expense of 20:1 ∆11 (data not shown).
Homozygous T3 lines were analyzed to examine the range of VLCFA proportional re-distribution induced
by expression of the nasturtium FAE gene. The 12 best T3 lines are shown in Figure 3.
Figure 3. Fatty acid composition of transgenic Arabidopsis seeds. Proportions of 20:1 c 11 and 22:1 c13 in seed oils from non-
transformed A. thaliana ecotype Wassilewskija (ntWS), 2 plasmid-only transgenic control lines (RD10-6 and RD15-8), and the twelve
best A. thaliana T3 homozygous transgenic lines expressing the T. majus FAE gene under control of the napin promoter.
The erucic acid content was increased by up to 7 to 8-fold in lines 15-2, 15-10, 16-1, 20-1 and 23-8. Small
increases in the proportions of 24:1 ∆15 were also observed. There was also a relatively significant
increase in the proportion of saturated VLCFAs. The content of 22:0 and 24:0 in the best T transgenic
lines increased up to 2.2 and 0.7% in comparison to 0.2 and 0.08% respectively, in the wild type
background, (data not shown).
Compared to other FAE genes heterologously-expressed in Arabidopsis the current expression of
the nasturtium FAE gene has resulted in the highest increase in erucic acid proportions observed thus far in
A. thaliana. For instance, introducing the jojoba FAE into Arabidopsis resulted in an increase in 22:1
proportions from about 2% in the control up to 4% in the transgenic seeds (Lassner et al., 1996). The
nasturtium FAE homolog described herein, may have a larger engineering impact when strongly expressed
in a seed-specific manner in H.E.A Brassicaceae.
Barret, P., Delourne, R., Renard, M., Domergue, F., Lessire, R., Delseny, M. and Roscoe, T.J. (1998) A rapeseed FAE1 gene is linked
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